title: Expression and secretion of functional recombinant μO-conotoxin MrVIB-His-tag in Escherichia coli.
abstract: μO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.
authors.affiliation: Key Lab of Tropical Biological Resources of Ministry of Education, Key Lab for Marine Drug of Haikou, Hainan University, Hainan, Haikou 570228, China.
authors.forename: Bingmiao
authors.initials: B
authors.lastname: Gao
authors.forename: Dongting
authors.initials: D
authors.lastname: Zhangsun
authors.forename: Yuanyan
authors.initials: Y
authors.lastname: Hu
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.forename: Lizi
authors.initials: L
authors.lastname: Sheng
authors.forename: Licong
authors.initials: L
authors.lastname: Fang
authors.forename: Xiaosa
authors.initials: X
authors.lastname: Wu
authors.forename: Jinpeng
authors.initials: J
authors.lastname: Yu
authors.forename: Sulan
authors.initials: S
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D052078:Conus Snail / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D020916:Conotoxins; D011993:Recombinant Fusion Proteins; C093339:conotoxin MrVIB
keywords: Affinity chromatography; Escherichia coli; PelB leader; Recombinant conotoxin; Secretory expression; μO-conotoxin MrVIB